Active nems arrays for biochemical analyses

ABSTRACT

A biofunctionalized nanoelectromechanical device (BioNEMS) for sensing single-molecules in solution by measuring the variation in the mechanical displacement of the BioNEMS device during a binding event is provided. The biofunctionalized nanoelectromechanical device according to the invention generally comprises a nanomechanical mechanical resonator, a detector integral with the mechanical resonator for measuring the mechanical displacement of the resonator, and electronics connected to the detector for communicating the results to a user. A system of biofunctionalized nanoelectromechanical devices and a method for utilizing the biofunctionalized nanoelectromechanical device of the present invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is based on U.S. Application No. 60/224,109, filed Aug.9, 2000, the disclosure of which is incorporated by reference.

FIELD OF THE INVENTION

This invention is generally directed to biofunctionalizednanoelectromechanical devices (BioNEMS) for enabling dynamicalsingle-molecule force assays of solutions.

BACKGROUND OF THE INVENTION

The revolution in molecular biology provided by DNA cloning andsequencing techniques, X-ray crystallography and NMR spectroscopy hasoffered unprecedented insights into the molecules that underlie the lifeprocess. However, in contrast to the dramatic rate of progress insequencing and structural approaches, there remain major stumblingblocks in applying modern molecular knowledge fully, as many of theanalytical techniques presently available remain remarkably similar tothose used in the relatively early days of molecular biology andbiochemistry.

For example, conventional gel electrophoresis and “blotting” techniquesfor determining the presence and amount of a given messenger RNA (mRNA)in a cell requires vast quantities of cells (˜10⁹), and 2 days tocomplete. Even the most advanced DNA array chip techniques require˜2×10⁷ cells. Accordingly, advances in fields ranging from molecularmedicine and basic cell biology to environmental toxicology are beinghampered by the bottleneck generated by the sensitivity and speed ofthese conventional analytical techniques.

A growing literature of chemical force microscopy (CFM) has shown that amodified Atomic Force Microscope (AFM) can be tailored to measure thebinding force of interactions ranging from single hydrogen bonds andsingle receptor-ligand interactions to single covalent bonds. Forexample, an early study showed the force required to break a singlehydrogen bond to be on the order of 10 pN and subsequent work enabledthe direct measurement of receptor/ligand interactions (˜50-250 pN) andDNA hybridization (˜65 pN-1.5 nN). CFM has also been utilized to studyconformational changes such as the deformation of the polysaccharidedextran by an applied force and have elucidated the unfolding of theprotein titan (˜100-300 pN). In addition to the above experimentsperformed with CFM, important advances have been made with opticaltweezers. In particular, they have been used to study step-wise forcesin biological motor motion and sub-pN polymer dynamics.

While the range of forces associated with many biochemical systems arewell within the capability of AFM instrumentation to detect, there aresevere limitations to the systems in which these devices can be used.For example, an AFM cantilever in solution does not have the temporalresponse characteristics needed to permit the binding and unbinding ofbiological ligands and their receptors to be followed reliably.Especially important are variation on the few μs timescale,characteristic of important classes of conformational changes in largebiomolecules. High frequency response is also critical to following thestochastic nature of receptor ligand interaction. Most receptor-ligandpairs interact dynamically: binding, remaining engaged for times rangingfrom microseconds to seconds (depending on the exact receptor-ligandpair), and then releasing. The analysis of biomolecules is thus limitedby both the vast quantities of materials required and the smearing intime inherent in even the most sensitive assays to date.

Perhaps even more significant is the substantial size of the equipmentrequired for performing AFM/CFM, and the density limits imposed byoptical detection of the probe motion. In addition, although the sensingmechanism is generally compact, even the so-called “lab on a chip”devices optical detectors are typically employed which require large,complicated support machinery, such as readers and sample preparationapparatus. These are not portable or easily reduced in size.

Third, optical tweezers employ diffraction-limited spots, hence theoptical gradient forces generated are far too spatially-extended topermit direct manipulation of individual biomolecules under study.Instead, biofunctionalized dielectric beads typically having diametersin the range 0.1 to 1 μm, are used to adhere to the analytes.Accordingly, this technology is not readily scalable to nanometerdimensions or to large-scale integration.

Finally, all of the aforementioned techniques involve force sensors withactive surface areas that are quite large compared to the molecularscale; hence it can be very difficult to achieve single-moleculesensing.

Accordingly, a need exists for a system and method for single moleculesensing in solution having higher sensitivity and temporal response withreduced overall size and active surface area.

SUMMARY OF THE INVENTION

The present invention is directed to a biofunctionalizednanoelectromechanical device (BioNEMS) for sensing single-molecules insolution. This can be accomplished in two distinct modes of operation.The first is “passive” and involves measuring the variation in theresonance motion of the BioNEMS device during a binding event. Thesecond is “active” and involves driving the devices with an externalsignal and looking for changes in the response upon a molecular bindingevent. The molecular detector according to the invention generallycomprises at least one nanomechanical resonator, a detector integralwith the mechanical resonator for measuring the vibration of theresonator, and electronics connected to the detector for communicatingthe results to a user.

In one embodiment, the molecular detector comprises a solution reservoirwhich contains the solution to be tested, a biofunctionalized mechanicalresonator arranged within the reservoir in fluid contact with thesolution, and a detector integral with the resonator for detecting theresonance of the resonator. During operation, the Brownian fluctuationsinherent in a non-turbulent solution drive random fluctuations in theposition of the mechanical resonator. The spectral density of thesolution-induced response will depend on the nature of the solution,i.e., viscosity, temperature, flow; and the geometry of and the materialused to construct the mechanical resonator. A molecule binding out ofsolution onto the surface of the resonator will inherently change themechanical properties of the resonator causing a variation in theresponse. The resonator is preferably biofunctionalized such that onlyspecified molecules will bind thereto, such that a binding eventindicates the presence of the specific molecule in the solution. Thedetector is engaged with the resonator to detect the response over timesuch that a change in the response can be measured to determine when abinding event occurs and multiple changes in the resonance can bemonitored to determine the frequency of binding events for a particularsample. The measurement of a resonance change can be used to determinethe absolute presence of a particular molecule in a solution, and thefrequency of binding events can be utilized to determine theconcentration of the molecule in a particular solution.

Any mechanical resonator or device suitable to provide mechanicalresponse in a solution may be utilized in the present invention, suchas, for example, vibrational resonators, counter rotating and rotatingresonators, torsional resonators, or compound resonators. Forsimplicity, all such putential mechanical detection devices will behereafter referred to as “resonators”. The resonator may be made fromany suitable material, such as, for example, silicon oxide, silicon,silicon carbide and gallium arsenide. The resonator may have anyphysical properties suitable for detection of single-molecular bindingevents in solution. For example, the resonator may have a thicknessbetween about 10 nm and 1 μm, a width between about 10 nm and 1 μm, anda length between about 1 μm and 10 μm. The resonator may have aresonance motion vacuum frequency between about 0.1 and 12 MHz. Theresonator may have a force constant between about 0.1 mN/m and 1 N/m.The resonator may have a Reynolds number between about 0.001 and 2.0.The resonator may have a mass loading coefficient between about 0.3 and11. Finally, the resonator may have a force sensitivity of about 8fN/√Hz or greater.

In one embodiment of the invention, the mechanical resonator is avibrating cantilever of simple or complex geometry. In such anembodiment, the cantilever is preferably a piezoresistive device suchthat the response is measured by sensing the voltage change in thecantilever over time. In such an embodiment, the molecular detector ispreferably biofunctionalized with a ligand or receptor.

In another embodiment, the molecular detector further comprises asubstrate disposed within the reservoir and adjacent to the resonator,where the substrate is biofunctionalized with a ligand capable ofmolecular interaction with the receptor, or vice-versa. Alternatively,the substrate may also be biofunctionalized with a receptor that is notcapable of molecular interaction with the receptor on the resonator, butwhich is capable of molecular interaction with a ligand which itself iscapable of molecular interaction with the receptor on the resonator.

In still another embodiment, the molecular detector comprises at leasttwo resonators arranged adjacent to one another, wherein one of theresonators is biofunctionalized with a receptor to form a receptorresonator and at least one of the resonators adjacent to the receptorresonator is biofunctionalized with a ligand capable of molecularinteraction with the receptor such that the resonators can be coupledthrough the ligand/receptor functionalization.

In yet another embodiment, the molecular detector comprises at least tworesonators arranged adjacent to one another, wherein at least one of theresonators is a driver resonator biofunctionalized with a receptor andhaving a driving element capable of resonating the driver resonator at achosen frequency or frequencies, and at least one of the resonatorsadjacent to the driver resonator is biofunctionalized with a ligandcapable of molecular interaction with the receptor on the driverresonator such that the resonators can be coupled through theligand/receptor functionalization.

In still yet another embodiment, the molecular detector comprises atleast three resonators, including, two driver resonators comprisingdriving elements capable of resonating the driver resonators at a chosenfrequency in antiphase to each other, and a follower resonator disposedbetween the two driver resonators. In such an embodiment, at least oneof the driver resonators is biofunctionalized with a receptor and thefollower resonator is biofunctionalized with a ligand capable ofmolecular interaction with the receptor on the driver resonator suchthat the resonators can be coupled through the ligand/receptorfunctionalization. In such an embodiment, the driver may be any devicesuitable for driving the resonator at a specified frequency, such as,for example, a piezoresistive driver device.

In still yet another embodiment, the detector is integral with theresonator. Any detector suitable for detecting the response of theresonator may be utilized, such as, for example, a piezoresistivetransducer or an optical detector. In an embodiment utilizing apiezoresistive transducer, the transducer may be made of p+ dopedsilicon.

In still yet another embodiment, the invention is directed to a systemof molecular detectors as described above. In one such embodiment themolecular detector system comprises at least one microfluidic channeland at least one array of molecular detector devices disposed within theat least one microfluidic channel, wherein the array comprises aplurality of biofunctionalized nanometer-scale mechanical resonators andwhere each resonator has at least one detector for measuring theresponse motion of the resonator.

In still yet another embodiment, the invention is directed to a methodof utilizing a molecular detector as described above. In one suchembodiment the method of detecting a molecule of interest comprisesproviding a molecular detector comprising a biofunctionalized nano-scaleresonator. Placing the molecular detector into a solution such that theresonator moves based on the thermal motion of the solution and suchthat in the presence of a species capable of molecular interaction withthe biofunctionalized resonator the response of the resonator isrestricted, and measuring the response of the resonator such that achange in the response of the resonator is communicated to a user.

In still yet another embodiment, the invention is directed to a methodof manufacturing a molecular detector as described above. In one suchembodiment the method of manufacturing the molecular detector comprisessupplying a substrate, depositing a photoresist on the substrate,exposing a pattern comprising the resonator on the photoresist, etchingthe substrate to form the resonator, and removing the photoresist.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bebetter understood by reference to the following detailed descriptionwhen considered in conjunction with the accompanying drawings wherein:

FIG. 1 is a schematic depiction of a first embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 2 is a schematic depiction of the operation of the first embodimentof a biofunctionalized nanoelectromechanical sensing device according tothe present invention.

FIG. 3 a is a schematic depiction of a second embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 3 b is a schematic depiction of a third embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 3 c is a schematic depiction of a fourth embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 3 d is a schematic depiction of a fifth embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 3 e is a schematic depiction of a sixth embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 3 f is a schematic depiction of a seventh embodiment of abiofunctionalized nanoelectromechanical sensing device according to thepresent invention.

FIG. 4 is a pictorial depiction of exemplary mechanical resonatorsaccording to the present invention.

FIG. 5 is a schematic diagram of a conventional surface-etchingtechnique for producing a biofunctionalized nanoelectromechanicalsensing device according to the present invention.

FIG. 6 is a pictorial depiction of a prototype of a biofunctionalizednanoelectromechanical sensing device according to an exemplaryembodiment of the present invention.

FIG. 7 is a graphical representation of the detection properties of aprototype of a biofunctionalized nanoelectromechanical sensing deviceaccording to the present invention.

FIG. 8 is a graphical representation of the detection properties of aprototype of a biofunctionalized nanoelectromechanical sensing deviceaccording to the present invention.

FIG. 9 is a graphical representation of the detection properties of aprototype of a biofunctionalized nanoelectromechanical sensing deviceaccording to the present invention.

FIG. 10 is a graphical representation of the detection properties of aprototype of a biofunctionalized nanoelectromechanical sensing deviceaccording to the present invention.

FIG. 11 is a graphical representation of the detection properties of aprototype of a biofunctionalized nanoelectromechanical sensing deviceaccording to the present invention.

FIG. 12 is a graphical representation of the detection properties of aprototype of a biofunctionalized nanoelectromechanical sensing deviceaccording to the present invention.

FIG. 13 is a schematic depiction of a second embodiment of a system ofbiofunctionalized nanoelectromechanical sensing devices according to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

A biofunctionalized nanoelectromechanical device (BioNEMS) capable ofsensing single-molecules in solution by measuring the variation in theresonance motion of a BioNEMS resonator device during a binding event isdescribed herein. The biofunctionalized nanoelectromechanical deviceaccording to the invention being henceforth referred to as a moleculardetector.

The molecular detector 10 according to one embodiment of the inventionis shown schematically in FIGS. 1 and 2 and comprises a solutionreservoir 12 containing a solution 14 having at least onebiofunctionalized nanoelectromechanical resonator 16 arranged therein. Adetector 18 in signal communication with an electronic signal processor20 is attached integrally to the resonator 16 such that any movement bythe resonator 16 is measured by the detector 18 amplified andtransmitted to the processor 20.

During operation, as shown in FIG. 2, the thermal fluctuations orBrownian motion inherent in the solution 14 create mechanicaldisplacement 22 of the position of the mechanical resonator 16, whilesimultaneously the presence of the solution 14 around the resonator 16produces a dampening force on the resonance motion of the resonator 16.In the case of the vibrational cantilever resonator 16 shown in FIGS. 1and 2, the Brownian movement of the molecules in the solution 14 createa mechanical displacement of the free end of the resonator 16. Thedynamic properties of this solution-induced displacement or response 22depends on the nature of the solution 14, i.e., viscosity, temperature,flow; and the geometry of and the material used to construct themechanical resonator 16. Although the thermal buffeting and solutiondampening of the resonator 16 makes conventional resonance detectiontechniques associated with AFM difficult to perform, molecules 24binding out of solution 14 onto the surface of the resonator 16 changethe mechanical properties of the resonator 16 causing a variation orrestriction in the thermally induced resonance 22 and this restrictionis then sensed by the detector 18 amplified and communicated to theprocessor 20. To ensure that the detector 18 only registers the presenceof specified molecules of interest, the surface of the resonator 16 maybe biofunctionalized or modified such that only specified molecules willbind thereto. For example, in FIGS. 1 and 2, the resonator 16 has beenbiofunctionalized with a ligand 26 chosen such that only a specifiedreceptor molecule 24 will bind thereto. Such a modification, allows forthe detection of minute quantities of specific molecules in the solution14 b utilizing the detector 10 according to the current invention.

Table 1, below displays a list of physical characteristics of a seriesof typical simple vibrational cantilever resonators according to FIGS. 1and 2.

TABLE 1 Characteristics of Simple Vibrational Cantilever Resonators Vac.Force Mass Thickness Width Length Freq. Constant Loading # (t) (w) (l)MHz (k) mN/m

Coeff. 1 100 nm   1 μm 10 μm  1.2 39 1.884 3.37 2 30 nm 300 nm 3 μm 4.112 0.5793 3.37 3 30 nm 100 nm 3 μm 4.1 3.9 0.0644 1.12 4 10 nm 300 nm 3μm 1.4 0.43 0.1978 10.11 5 10 nm 100 nm 3 μm 1.4 0.14 0.0220 3.37 6 10nm 100 nm 1 μm 12 3.9 0.1884 3.37 7 10 nm  30 nm 1 μm 12 1.2 0.0170 1.018 10 nm  10 nm 1 μm 12 0.40 0.0019 0.34

Although a simple single resonator 16 single ligand biofunctionalized 26detector 10 is shown in FIGS. 1 and 2, any combination of resonators 16and biofunctionalization can be utilized to create detectors 10 havingunique assay properties. Examples of some exemplary molecular detectors10 according to the current invention are shown in FIGS. 3 a to 3 f, anddiscussed below.

FIG. 3 a shows a molecular detector 10 comprising a single resonator 16with a ligand biofunctionalization 26′ and a substrate 28 with areceptor biofunctionalization 26″ designed to assay for either thepresence of a free receptor or free ligand in solution or to assay forcompounds that stabilize or compete with the interaction between thefunctional ligand/receptor. As shown, the resonator 16 will be tetheredto the substrate 28 when the ligand 26′ and receptor 26″ interact suchthat the mechanical response 22 of the resonator 16 is stronglyrestricted.

FIG. 3 b shows a molecular detector 10 comprising a single resonator 16with a receptor biofunctionalization 26′ and a substrate 28 with asecond receptor biofunctionalization 26″ designed to assay for molecules24 that contain target recognition sites for both receptors 26′ and 26″on the same molecule.

FIG. 3 c shows a molecular detector 10 comprising multiple resonators 16with a simple receptor biofunctionalization 26 designed to assay forsingle molecules 24, in which the ligand molecules 24 in the solution 14have been modified with star dendromers 30 such that the binding of theligand molecule 24 to the receptor biofunctionalization 26 more greatlyalters the viscous drag, and therefore the mechanical response 22 of theresonator 16. Although star dendromer modifiers 30 are shown in thisembodiment, any modifier which would enhance the resonator/solutioncoupling to provide sensitivity enhancement to the molecular detector 10may also be utilized.

FIG. 3 d shows a molecular detector 10 comprising multiple coupledresonators 16 with a receptor biofunctionalization 26′ on one resonator16′ and a ligand biofunctionalization 26″ on an adjacent resonator 16″such that the motion of the resonators 16′ and 16″ is coupled throughthe ligand/receptor biofunctionalization and such that the motion ofboth resonators is monitored simultaneously. In this embodiment, thecorrelation of the motion of the two resonators 16′ and 16″ allows forgreater noise reduction, increasing the sensitivity of the moleculardetector 10. This molecular detector 10 could be designed to assay forcompounds that either bind with or stabilize or compete with thefunctional ligand/receptor interactions between the adjacent resonators.

FIG. 3 e shows a molecular detector 10 comprising at least two differentresonators: a driver resonator 16 a and a follower resonator 16 b. As inthe embodiment shown in FIG. 3 d, a receptor biofunctionalization 26′ isprovided on the driver resonator 16 a and a ligand biofunctionalization26″ is provided on the adjacent follower resonator 16 b such that themotion of the resonators 16 a and 16 b is coupled through theligand/receptor biofunctionalization and such that the motion of bothresonators 16 a and 16 b is monitored simultaneously. However, in theembodiment shown in FIG. 3 e a driver (not shown), actuatedpiezoelectrically, thermoelastically or by other physical mechanisms,actively drives the motion of the driver resonator 16 a such that themotion 22 is tuned to the most sensitive amplitude and frequencypossible for the geometry of the driver resonator 16 a. The correlatedmotion of the driver resonator 16 a and follower resonator 16 b are thenmonitored to detect whether the ligand/receptor pair are functionallylinked. A molecular detector 10 of this design could then be utilized toassay for compounds that either bind with or stabilize or compete withthe functional ligand/receptor interactions between the adjacentresonators.

FIG. 3 f shows a molecular detector 10 comprising at least threedifferent resonators: a (+) driver resonator 16 a, a (−) driverresonator 16 b and a follower resonator 16 c. As in the embodiment shownin FIG. 3 e, a receptor biofunctionalization 26′ is provided on one ofthe driver resonators 16 a and a ligand biofunctionalization 26″ on theadjacent follower resonator 16 c such that the motion of the resonators16 a and 16 c is coupled through the ligand/receptorbiofunctionalization and such that the motion of both resonators 16 aand 16 c is simultaneously monitored. As in the embodiment shown in FIG.3 e a piezoelectric driver (not shown) actively drives the resonancemotion of the driver resonators 16 a and 16 b such that the motion istuned to the most sensitive amplitude and frequency possible for theresonator geometry. The correlated motion of the driver resonator 16 aand follower resonator 16 c are then monitored to detect whether theligand/receptor pair are functionally linked. However, in the activelydriven embodiment shown in FIG. 3 e, hydrodynamic coupling between theresonators 16 a and 16 c may limit the dynamic range of the moleculardetector 10. Providing a second active resonator 16 b, operated inantiphase, nulls the hydrodynamic coupling, thereby improving thesignal/noise of the molecular detector 10 thus produced. A moleculardetector 10 of this design could then be utilized to assay for compoundsthat either bind with or stabilize or compete with the functionalligand/receptor interactions between the adjacent resonators. There maybe advantages to configuring multiple-driver geometries (beyond the pairof drivers described here) to provide more refined schemes for nullingthe background fluidic coupling to the “detector” cantilever.

Although the embodiments of the molecular detectors 10 discussed abovein relation to FIGS. 1 to 3 all describe a single moleculeligand/receptor biofunctionalization 26, it will be understood that anysuitable biofunctionalization 26 may be utilized in the currentinvention, such as, DNA hybridization, chemical bonds and proteinunfolding. For example, the molecular detector may by biofunctionalizedto screen the products of combinatorial chemistry, or to profile geneexpression in cells, or to sense the concentrations of growth factors,hormones and intracellular messengers in cell biology, or to yieldinformation about specific blood chemistry, or as a general physiologysensor, or as a detector for exposure to pathogens or toxins either inthe environment or in a patient. Likewise, although all of the exemplaryembodiments shown in FIGS. 1 to 3 all show single biofunctionalizedsites 26 on the resonators 16, any method of biofunctionalization ornumber of biofunctionalized sites may be utilized on the resonators 16of the current invention.

Although the embodiments of the resonator 16, shown in FIGS. 1 to 3 areall depicted as simple vibrational cantilever resonators 16, it shouldbe understood that any NEMS construct capable of resonance motion underthe thermal or Brownian motion of the solution 14, wherein the resonanceis sufficiently sensitive to allow detection of a restriction in theresonance motion 22 caused by a single molecule binding event can beutilized in the present invention. FIG. 4 shows pictorialrepresentations of several different conventional NEMS resonators 16suitable for use in the current invention, such as, for example,rotational resonators, torsional resonators and composite resonators. Inaddition, it should be understood that although the resonators describedabove are all macrodevices, resonators comprising single moleculescoupled to a substrate may be utilized according to the presentinvention such that the molecule itself would be modified to interactwith a molecule of choice in a solution.

The present invention is also directed to a method of manufacturing theBioNEMS molecular detector 10. FIG. 5, shows a schematic diagram of anexemplary technique for manufacturing a BioNEMS resonator 16 accordingto the present invention utilizing surface-etching. There are two partsto manufacturing the resonator 16 of the present invention utilizing aNEMS manufacturing method; the actual manufacturing process, and themask design. FIG. 5, shows one embodiment of the method for making theresonator 16 according to the present invention, including the number ofphotolithographic steps required, and how the resonator 16 is separatedfrom the substrate. The basic sequence, as shown, include: (a) examiningand cleaning a starting substrate comprising, in the embodiment shown,three layers, a structural layer 32, a sacrificial layer 34 and asubstrate layer 36; (b) modifying the surface of the structural layer 32to form the resonator 16 via an electron beam mask 38 and depositing thephotoresist and pattern resist etch metal for the resonator 16; (c)etching the pattern into the structural and sacrificial layers 32 and34; and (d) etching the sacrificial layer 34 to undercut the resonator16 to free the resonator 16. Although this embodiment only shows anetching process which undercuts the sacrificial layer 34, it should beunderstood that additional etching may be performed to create deeperundercuts and/or etching of the substrate 36 below such that insulationbetween the resonator 16 and the substrate 36 is increased.

While the above embodiment exemplifies a method for forming theresonator 14 of the present invention utilizing a conventional NEMSprocess, any manufacturing process suitable for forming the nanometerresonator 16, such as, for example, wafer bonding and etch-back may beutilized. In the wafer bonding and etch-back process a silicon wafersubstrate has a very thick oxide layer deposited or thermally grown onthe surface. This thick oxide layer is then covered by a thin siliconnitride layer. The resonator 16 is deposited and fabricated on thissilicon nitride layer. The surface of the resonator 16 is then coveredby resist, and the back of the substrate 36 is removed chemicallyleaving only a “frame” to support the devices. When utilizing thisapproach, the resonator 16 is preferably not close to the substrate 36.

The resonator 16 can be fabricated utilizing any suitable substratematerial, such as, for example, silicon. In a preferred embodiment, asingle-crystal silicon substrate is utilized for the resonator 16. Othersilicon materials may also be utilized to make the resonator 16 of thepresent invention, such as, for example, thick epitaxial silicon onsingle crystal wafers with highly doped layers as leads, orpolycrystalline silicon. Although the manufacturing process describedabove describes the surface nanomachining of a silicon-based material,the resonator 16 of the current invention can be made of any materialsuitable for surface nanomachining, capable of biofunctionalization andinert to chemical modification by and of the molecules 24 in thesolution 14. Examples of conventional nanomachining materials suitablefor use in the current invention include: silicon-based systems, such assilicon oxide (SOI) or silicon carbide and gallium-arsenide-basedsystems (GaAs). Other substrate materials may be used, as well,including insulating materials such as diamond and quartz thin films.

Any detector 18 suitable for detecting the resonance motion of theresonator 16 in solution may be utilized in the molecular detector 10 ofthe current invention. For example, the detector 18 may comprisevibrational or strain sensitive devices integrally connected to theresonator 16, as shown in FIGS. 1 and 2. In one exemplary embodiment thedetector 18 is a piezoresistive strain transducer, as shown in FIG. 1.In this embodiment the transducer detector 18 converts the motion of theresonator 16 into an electrical signal via the strain-induced change inresistance of a conducting path on the top surface of the resonator 16.These resistance changes are then amplified and communicated to aprocessor 20 designed to provide a read-out of the signal changes.Although the detector 18 may be made of any suitable material, in oneembodiment it is made from a p+ doped silicon epilayer formed on the topsurface of the resonator 16.

Although only strain-type transducer detectors are described above, anydetector suitable to monitor the motion of the resonator 16 on atime-scale suitable for monitoring the biomolecular interactions ofinterest may be utilized. For example, the detector 18 may also comprisean externally mounted device, such as, an optical-laser, fluorescencebased position sensor, electromagnetic or magnetic.

The signal monitor system and processor 20 for any of the abovedetection schemes can comprise any suitable digital signal processorcapable of measuring the signal change from the detector 18 andtransmitting that information to the user, such as, for example, aprinted circuit board having a pre-amplifier, an AD converter and drivercircuit, and a programmable chip for instrumentation specific software;or a multichip module comprising those elements.

Regardless of the specific embodiment of the molecular detector 10utilized, all operate on the principle that a BioNEMS resonator willinherently posses a large thermally driven motion or mechanical responsewhen disposed within a solution due to the repeated interaction betweenthe resonator and the molecules of the solution, and that a chemicalbond between the functionalized portion of the resonator and themolecule of interest will produce a detectable alteration of themechanical response.

FIG. 6 shows a prototype notched cantilever resonator 16 utilized totest the sensitivity of molecular detectors 10 made according to thepresent invention. First, the theoretical force sensitivity of themolecular detector 10 was calculated and then the actual performance ofa series of detectors utilizing the resonator shown in FIG. 6 wastested.

Table 2, below, summarizes the physical parameters for threeprototypical notched cantilever resonator 16 according to FIG. 6.Utilizing the cantilever resonator prototypes listed in Table 2 thephysical properties of the molecular detector of the current inventionwere calculated.

TABLE 2 Characteristics of Notched Vibrational Cantilever Resonators #(t) (w) (l) (l₁) (b) ω₀/2π K 1 130 nm  2.5 μm 15 μm 2.5 μm  0.6 μm 0.51MHz  34 mN/m 2 130 nm 300 nm 10 μm 2.0 μm 100 nm  1.3 MHz  20 mN/m 3  30nm 100 nm  3 μm 0.6 μm  33 nm  3.4 MHz 3.0 mN/m

Because the resonator 16 is large compared to the size of the molecules24 in the solution 14, the thermal motion of the resonator 16 insolution 14 may be modeled in terms of stochastic forces, which areMarkovian (because the time scale of the molecular collisions with theresonator are short compared to the frequencies of the macroscopicresonance motion of the resonator), and Gaussian (because themacroscopic motion is formed by a large number of molecular collisions).Accordingly, the resonance motion of the resonator 16 in the solution14, in its fundamental mode, can be described and modeled by thefluctuation-dissipation theorem.

Any suitable calculation can be utilized to estimate this dissipation,such as, simplified geometric model estimations, low Reynolds numberfluid solution calculations, or experimental measurements. Thestochastic motion (x) of the resonator 16 may then be found by solvingits dynamical equation with an additional fluctuating force with thespectral density. For resonators at the submicron scale in solution, asin the present invention, dissipation is dominated by the viscous motionof the fluid driven by the vibration of the resonator 16.

Because the size of the resonator 16 is much larger than the size of theindividual molecules 24 in the solution 14 colliding therewith, anapproximation of the force on each small section of the resonator 16 asa result of the solution 14 impinging thereon is equal to the force ofthe solution 14 acting on the length of an infinite beam with the samecross-section and velocity.

In the example of a single rectangular vibrational cantilever resonator16 as shown in FIG. 1, the loading of the resonator 16 can beapproximated by the Stokes equation for a cylinder according to EQ. 1,below.

$\begin{matrix}{{L(\omega)} = {\frac{{\pi\rho}_{L}w^{2}}{4}{\Gamma ()}}} & (1)\end{matrix}$

where the prefactor is simply the volume displaced by the resonator 16,while the function Γ, which depends solely on the Reynolds number (

), must be calculated from the motion of the solution 14. In thisapproximation, the fluidic forces from the solution 14 at each frequencyand on each section of the resonator 14 are proportional to thedisplacement at that point.

Alternatively, a more complete calculation of the resonance motion of aresonator can be made utilizing the basic equations of motion. In thecase of a notched vibrating cantilever resonator 16, as shown in FIG. 6,the equation of motion for the displacement (x) at the end of theresonator 16 is that of a simple vibrating cantilever in vacuumaccording to:

$\begin{matrix}{{\overset{\sim}{x}} = \frac{\overset{\sim}{F}}{\left\{ {\left\lbrack {K - {\omega^{2}{M_{eff}(\omega)}}} \right\rbrack^{2} + {\omega^{2}{\gamma_{eff}^{2}(\omega)}}} \right\}^{1/2}}} & (2)\end{matrix}$

where x describes the motion of the free end of the cantilever resonator16, F is the applied force, K is a force constant dependent on thegeometry of a resonator 16 of width (w), thickness (t) and length (l).EQ. 2 provides a complete description of the resonator's 16 resonanceresponse both to the externally applied forces and, through thefluctuation-dissipation theorem, to the stochastic forces imparted fromthe solution 14.

For a notched cantilever, as shown in FIG. 6, the force constant couldbe found according to the equation:

$\begin{matrix}{K = \frac{{Et}^{3}}{{4{l^{3}/w}} + {\left( {{2l_{1}^{3}} - {6{ll}_{1}^{2}} + {6l^{2}l_{1}}} \right)\left( {\frac{1}{b} - \frac{2}{w}} \right)}}} & (3)\end{matrix}$

where (w) is the width of the end of the resonator 16, (l) is the lengthof the resonator 16, (t) is the thickness of the resonator 16, (b) isthe width of the notch legs 30 of the resonator 16, and (l₁) is thelength of the notched portion 32 of the resonator 16.

The equations of motion for the resonator 16 are complicated because ofthe presence of a dynamic solution 14 surrounding and influencing themotion of the resonator 16. Accordingly, in solution M_(eff) is theeffective mass of the cantilever resonator 16, which is dependent on thefluid loading of the solution 14. In vacuum the effective mass followsthe equation:

$\begin{matrix}{M_{eff} \cong {{\alpha\rho}_{c}{{wtl}\left\lbrack {1 + {\frac{\pi}{4}T\; {Re}\left\{ \Gamma \right\}}} \right\rbrack}}} & (4)\end{matrix}$

which itself is dependent on the fluidic mass loading coefficient Taccording to:

T=αρ _(L) w/(ρ_(c) t)  (5)

with ρL, ρC the density of the solution and resonator, respectively. Asa result, thin resonators experience relatively large fluid loading(where ρ_(L)/ρ_(C)=2, T ranges from 1 to 5). The value of Re{Γ} is unityfor large

, is around 4 at

equals 1, and continues to increase as

decreases. Hence, for a value of w/t equal to 2, the mass loading factoris at least 5 at

equal 1, and increases for proportionally thinner beams and lowerReynolds numbers.

In turn, γ_(eff) is the effective fluidic damping coefficient, accordingto EQ. 5, below.

$\begin{matrix}{\gamma_{eff} \cong {\alpha \; \frac{{\pi\rho}_{L}}{4}w^{2}{l\left\lbrack {\omega \; {Im}\left\{ \Gamma \right\}} \right\rbrack}}} & (6)\end{matrix}$

The parameter α relates the mean square displacement along the beam tothe displacement at its end. For the fundamental mode of a simplerectangular vibrational cantilever resonator 16, as shown in FIG. 1,α=0.243. In comparison, the notched vibrational cantilever resonator 16,shown in FIG. 6, α=0.333.

In addition, the term Γ corresponds to the fluidic coupling between theresonator 16 and the solution fluid 14 according to:

$\begin{matrix}{{\Gamma ()} = {1 + \frac{4{{iK}_{1}\left( {{- i}\sqrt{i\; }} \right)}}{\sqrt{i\; \; {K_{0}\left( {{- i}\sqrt{i\; }} \right)}}}}} & (7)\end{matrix}$

where the Reynolds number (

) is given by the equation:

(ω)=ωw ²/(4v)  (8)

where v is the kinematic viscosity of water and is equal to 1.022×10⁻⁶m²/s at 293 K.

Accordingly, for frequencies below ˜1 MHz with resonators having a widthless than or equal to 1 μm, the Reynolds number is less than or equal to1.6. Thus, the damping of the resonator 16 arising from the motion ofthe solution 14 fluid is most dependent on the dimensions of theresonator 16 transverse to the resonance motion, e.g., in the case of avibrational cantilever as shown in FIG. 1, the width and length of theresonator. This analysis indicates that with uniform scaling down of alldimensions, w, t, l∝d, the damping of a resonator 16 in solution 14decreases as d with decreasing size of the resonator 16, increasing thesensitivity of the molecular detector 10.

In Table 3, below, a list of the calculated properties of the prototypenotched vibrational cantilever resonators 16, as shown in FIG. 6, areprovided.

TABLE 3 Characteristics of Notched Vibrational Cantilever Resonators t wl l₁ (b) ω₀/2π K # (nm) (nm) (μm) (μm) (nm) (MHz) (mN/m)

T 1 130 2,500 15 2.5 0.6 0.51 34 5.0 8.22 2 130 300 10 2.0 100 1.3 200.19 0.986 3 30 100 3 0.6 33 3.4 3.0 0.054 1.42

As described above, the thermal noise component arises, as described bythe fluctuation-dissipation theorem, from the fluidic damping of thecantilever. The mechanical Q of these structures is approximated usingthe equation:

$\begin{matrix}{\left. Q \right.\sim\left. \frac{\omega \; M_{eff}}{\gamma_{eff}} \right.\sim\frac{{Re}\left\{ {\Gamma ()} \right\}}{{Im}\left\{ {\Gamma ()} \right\}}} & (9)\end{matrix}$

where fluid mass is assumed to dominate. It will be recognized that thisexpression is mostly independent of frequency, varying only over therange 0.2<Q<0.9 as the Reynolds number (

) changes from 10⁻³ to 1. As described above, and as expected from thecalculations, the mechanical Q of these resonators 16 in the solution 14is much less than 1, whereas their W's in vacuum are typically of on theorder of 10⁴. Hence the fluidic dissipation resulting from thesurrounding solution 14 completely determines the resonance 22 of theresonator 16.

To quantitatively determine the effective force sensitivity of theresonator 16 and ultimately the molecular detector 10 described by theabove equations of motion, the force acting on the resonator 16 from thethermal or Brownian motion of the solution 14 must be taken intoaccount. With this regard, the minimum detectable force is definedaccording to:

F _(min)(ω/ω₀)=[S _(F)(ω/ω₀)]^(1/2)  (10)

where the minimum detectable force (F_(min)) is defined by the force(S_(F)) acting on the resonator 16 as the result of the molecular motionof the molecules in solution 14. This stochastic force acting on theresonator 16 can be directly related to the dissipative coefficientappearing in EQ. 2, such that the force spectral density is given by theNyquist formula:

S _(F)=4k _(B) Tγ _(eff)  (11)

where k_(B) is Boltzmann's constant and T is the temperature of thesolution 14.

Likewise, the displacement fluctuations (S_(x)) are defined by themechanical responsivity to the spectral force (S_(F)), according to:

S _(x) ^((γ))(ω)=S _(F) ^((γ))(ω)R _(mech) ^(s)(ω)  (12)

where the mechanical responsivity R_(mech) having units m/N is definedaccording to EQ. 13, below.

R _(mech)=√{square root over (R(ω/ω₀))}/K  (13)

where R(ω/ω₀) is provided in analogy with Hooke's Law, −1/K=x/F:

$\begin{matrix}\begin{matrix}{{R\left( {f/f_{0}} \right)} \equiv \frac{K^{2}{\overset{\_}{x}}^{2}}{{\overset{\sim}{F}}^{2}}} \\{= \begin{bmatrix}{\left\{ {{\frac{\omega^{2}}{4\omega_{0}^{2}}\left( {1 + {\frac{\pi}{4}\overset{\_}{T}{Re}\left\{ {\Gamma \left\lbrack {\left( {\frac{\omega}{\omega_{0}}R_{0}} \right)} \right\rbrack} \right\}}} \right)} - 1} \right\}^{2} +} \\\left( {\frac{\pi}{16}\overset{\_}{T}\frac{\omega^{2}}{\omega_{o}^{2}}{Im}\left\{ {\Gamma \left\lbrack {\left( {\frac{\omega}{\omega_{o}}R_{0}} \right)} \right\rbrack} \right\}} \right)^{2}\end{bmatrix}^{- 1}}\end{matrix} & (14)\end{matrix}$

In FIG. 7, the response function R(ω/ω₀), for three differentvibrational cantilever geometries is provided. It is apparent from theplot that a finite frequency peak is present in the response function ofthe solution damped vibrational cantilever resonators.

As described in the previous section, the frequency dependentdisplacement spectral density and mean square response functionsobtained in the presence of fluid coupling allow an estimation of theforce sensitivity attainable for different resonator geometries.However, to determine the effective force sensitivity for the moleculardetector 10 according to the present invention it is also necessary todetermine the noise induced by the detector 18 or the electrical noiseof the system. In the three notched-vibrational cantilever resonatormolecular detector prototypes 10 shown in FIG. 6 and described above, astrain sensitive piezoelectric transducer 18 was utilized to detect theresonance motion of the resonator 16. Accordingly, three additionalterms are added to the real system force noise equation according to EQ.15, below.

$\begin{matrix}{\left\lbrack S_{F} \right\rbrack_{eff} = {\frac{1}{R_{mech}^{2}}\left\{ {\left\lbrack S_{x} \right\rbrack_{fluidic} + {\frac{1}{R_{detector}^{2}}\left( {\left\lbrack S_{V}^{out} \right\rbrack_{detector}^{RTO} + \left\lbrack S_{V}^{A} \right\rbrack_{amplifier}^{RTI}} \right)}} \right\}}} & (15)\end{matrix}$

In this equation S_(F) is equivalent to the spectral force or the forcefluctuations applied to the resonator 16, S_(X) is equal to thefluid-coupled noise of the resonator 16, S_(V) ^(out) is equal to thenoise generated by the detector 18, and S_(VA) is equal to the noisegenerated by the amplifier and other processor electronics 20.

In the case of the prototype S_(V) ^(out) arises from the thermal noiseof the piezoresistive transducer where S_(V) ^(out) is equal to:

S _(VA)=4k _(B) TR _(T)  (16)

while S_(VA) arises from the readout amplifier's voltage and currentnoise according to:

S _(VA) =S _(V) +S ₁ R _(T) ²  (17)

where S_(V) and S₁ are the spectral density of the amplifier's voltageand current noise respectively.

In those cases where the response extends down to low frequencies, athird term must also be considered, the 1/f noise (S_(1/f)) in thetransducer. Although this term must be considered, there is afundamental difference between the 1/f noise and that of thefluid-induce displacement fluctuations. As such, in a preferredembodiment a lock-in detection scheme is used to measure the resistancesuch that only the portion of the 1/f spectrum within the detectionwindow will contribute to the noise. Alternatively, by probing theresistance at frequencies above the 1/f knee, this source of noise canbe practically eliminated.

In contrast, the fluid-induced displacement fluctuation noise leads tochanges in the resistance of the resonator that are within thedetectable range regardless of the frequency probe current used. Hence,the entire noise spectrum from dc up to the frequency of the low passfilter is relevant.

The force sensitivity of the molecular detector 10 of the currentinvention, then, hinges on the maximum level of current bias that istolerable, given that the responsivity is proportional to the biascurrent (R=IG), where the gauge factor (G) is equal to:

$\begin{matrix}{G = {\frac{\partial R_{T}}{\partial x} = \frac{3{{\beta\pi}_{l}\left( {{2l} - l_{1}} \right)}R_{T}}{2{bt}^{2}}}} & (18)\end{matrix}$

and where the parameter π₁ is the piezoresistive coefficient of the p+transducer material. The factor β accounts for the decrease in G due tothe finite thickness of the of the conducting layer; β approaches unityas the carriers become confined to a surface layer of infinitesimalthickness.

To quantify some of the parameters for the prototype notched vibrationalcantilever resonators shown in FIG. 6, the resonance motion andresistance of the resonators was measured. FIG. 8 shows the measuredroom temperature fundamental resonance motion for the first prototypecantilever resonator listed in Table 2 in vacuum. FIG. 9 shows a plot ofthe displacement of the prototype cantilever shown in FIG. 6 caused bythe resonance motion versus resistance.

These plots yield a direct measurement of G=3×10⁷. For epilayers such asthose used in the prototype molecular detectors shown in FIG. 6, the EQ.18 yields a calculated value of β=0.7 and G=6×10⁸ Ω/m. For thetransducer geometry pictured in FIG. 6, a two-terminal (equilibrium)resistance of R_(T)=15.6 kΩ is obtained.

Using the values for the resistance and the gauge factor (G) above, itis possible to determine the maximum current bias, which is found bydetermining the maximum temperature rise deemed acceptable for thebiofunctionalization disposed along the resonator. The geometry of theprototype devices shown in FIG. 6 causes dissipation to occurpredominantly within the constriction regions (of width b). A roughestimate of the heat loss to the surrounding solution may be obtainedthrough the relationship:

$\begin{matrix}{{\kappa_{Si}A\frac{\partial^{2}T}{\partial x^{2}}} = {\kappa_{H_{2}O}P{\nabla_{n}T}}} & (19)\end{matrix}$

where P is the perimeter around cross-sectional area A of the resonator.Estimating that:

∇_(n) ˜T/w  (20)

and that,

$\begin{matrix}{\left. \frac{\partial^{2}T}{\partial x^{2}} \right.\sim\frac{2\left( {w + t} \right)\kappa_{H_{2}O}}{\kappa_{Si}{tw}^{2}}} & (21)\end{matrix}$

where κ_(Si)=1.48×10² W/mK is the thermal conductivity of silicon andκH2O=0.607 W/mK is the thermal conductivity of water. In the dissipativeregion x<l₁,

$\begin{matrix}{{2\kappa_{Si}{tb}{\left. \frac{\partial^{2}T}{\partial x^{2}} \right.\sim{- I^{2}}}R} + {\left( {b + t} \right)\frac{T}{b}\kappa_{H_{2}O}}} & (22)\end{matrix}$

where as boundary conditions, the temperature is continuous at l₁, as isthe heat flux; and δT/δx=0 at x=1.

This simple thermal conductance calculation indicates that, for example,a 1 K rise a the biofunctionalized tip is attained with a steady-statebias current of 250 μA, leading to a power dissipation of roughly 10670μW. The maximal temperature rise of 12K occurs within the constrictedregion, approximately 2.3 μm from the support. For this bias current,the prototype molecular detector 10 yields a responsivity of R=IG˜8μV/nm.

Utilizing these parameters, an estimated coupled force sensitivity canbe determined. For cantilever 1, assuming that a 1K rise at the tip istolerable, the transducer-induced displacement noise is found to be√S_(VT)/R=1.8×10⁻¹² m/√Hz. For a typical low noise readout amplifierwith voltage and current noise levels of ˜4 nV/√Hz and ˜5 fA/√Hz,respectively (typical for JFET input low noise amplifiers) these sameparameters yield an amplifier term √S_(VA)/R=4.4×10⁻¹³ m/√Hz.

To demonstrate the effects of scaling the resonator downward in size,cantilever resonators 2 and 3, having a geometry identical to that ofcantilever resonator 1, are also considered. Utilizing the physicaldimensions of cantilever 2 the above equations yields an R_(T)=67 kΩ anda G=7.4×10₉ Ω/m. For cantilever resonator 2, assuming an 0.05Ktemperature rise at the tip of the resonator is tolerable yields atransducer-induced displacement noise √S_(VT)/R=6.3×10⁻¹⁴ m/√Hz and areadout amplifier contribution of √S_(VA)/R=80×10⁻¹⁵ m/√Hz. Forcantilever resonator 3, the above equations yields an R_(T)=258 kΩ and aG=7.39×10₁₀ Ω/m. Again assuming an 0.05K temperature rise at the tip ofthe resonator is tolerable yields a transducer-induced displacementnoise √S_(VT)/R=3.8×10⁻¹⁴ m/√Hz and a readout amplifier contribution of√S_(VA)/R=3.3×10⁻¹⁵ m/√Hz.

In FIGS. 10 to 12 the coupled force sensitivity per unit bandwidthcalculations for the three prototype notched vibrational cantileverresonators 1 to 3 in Tables 2 and 3 utilizing three different detectorbias currents are plotted verse the thermal force noise of the solution.These calculations include the combined noise from fluidic, transducer,and readout amplifier sources.

FIG. 10 shows that for a temperature rise of 1K at the resonator tip,even the largest resonator (cantilever 1) yields a remarkably lowcoupled force sensitivity [S_(f) ^((c))]^(1/2)≦85 fN/√Hz for frequenciesbelow 100 KHz. This indicates that a molecular detector utilizing thecantilever 1 resonator would be capable of taking dynamical measurementson the ˜10 μs scale for absolute forces on the level of <30 pN withoutaveraging.

FIG. 11, shows that for an 0.05K temperature rise at the tip of theresonator the cantilever 2 resonator device yields even better forcesensitivity, [S_(f) ^((c))]^(1/2)≦20 fN/√Hz for frequencies below 0.5MHz (10% above the fluidic fluctuation limit). This indicates that amolecular detector utilizing the cantilever 2 resonator would be capableof taking dynamical measurements on the ˜2 μs scale for absolute forceson the level of <15 pN without averaging.

Finally, FIG. 12, shows the attainable force sensitivity for a deviceutilizing a cantilever 3 resonator. Again, for an 0.05K temperature riseat the tip of the resonator the cantilever 3 resonator device yields aforce sensitivity of [S_(f) ^((c))]^(1/2)≦10 fN/√Hz for frequenciesbelow 2 MHz (10% above the fluidic fluctuation limit) and the forcesensitivity rises to just ˜11 fN/√Hz for frequencies ≦3 MHz. Thisindicates that a molecular detector utilizing the cantilever 2 resonatorwould be capable of taking dynamical measurements on the ˜300 ns scalefor absolute forces on the level of <20 pN without averaging.

Accordingly, the achievable coupled sensitivity for the moleculardetector described herein, as low as ˜8 fN/√Hz, is limited predominantlyby the fluidic fluctuations of the solution. As shown in Table 4, below,this threshold detection limit is well below the interaction forces ofinterest in most biological and chemical processes.

TABLE 4 Interaction Forces Nature of Interaction Interaction ForceReceptor/Ligand Interaction 50-250 pN Avidin-Biotin 90-260 pNAntibody-Antigen 50-300 pn Cadherin-Cadherin 35-55 pN DNA Hybridization65 pN-1.5 nN Chemical Bond 1-10 nN Covalent (C—C, C—O, C—N) 4.0-4.5 nNCovalent (Au—S, Si—C) 1-3 nN H-bond 10 pN Unfolding Forces 100-300 pNProtein (Titin) unfolding 150-300 pN Dexran bond twists 100-300 pN

Although only molecular detectors 10 having single resonator assemblies16 are shown in the Figures and discussed in the text above, themolecular detector 10 according to the present invention may alsocomprise a large array or system of resonator assemblies. One exemplaryembodiment of such a system is shown schematically in FIG. 13, whichshows a multiple channel array 40 of molecular detectors 10, in whichthe array channels 42 are aligned in parallel on a single substrate 44such that multiple or parallel processing of molecular samples can becarried out at one time. In this embodiment, multiple moleculardetectors 10 are utilized for analysis of the molecules. It should beunderstood that while parallel and single array channels 42 are shown inFIG. 13, any suitable alternative geometry of channels 42 may beutilized such as, for example, folded channels may be used to increasethe length of the detector path without increasing the size of the arraybody 40. Although the embodiment shown in FIG. 13 discloses amulti-channel array 40 in which the detector channels 42 are separatedby walls 46, the multi-channel detector array 40 could alternativelycomprise a single “sheet” of detector arrays without walls between thechannels 42.

Further, while all of the resonators 16 of the molecular detector arraysystem 40 could be functionalized to monitor for a single substance, asdescribed in the previous embodiments, thereby providing greatlyenhanced detector sensitivity, the resonators 16 of the detector array40 system shown in FIG. 13 may also comprise individuallybiofunctionalized resonators such that multiple substances can beidentified and monitored simultaneously. In addition, any combination ofthe various resonator embodiments shown and discussed in relation toFIGS. 3 a to 3 f, above, may be utilized in the molecular detector arraysystem of the present invention.

Although specific embodiments are disclosed herein, it is expected thatpersons skilled in the art can and will design alternative moleculardetectors, methods to produce the molecular detectors and/or moleculardetector systems that are within the scope of the following claimseither literally or under the Doctrine of Equivalents.

1-32. (canceled)
 33. A molecular detector system comprising: at leastone microfluidic channel; at least one array of molecular detectordevices disposed within the at least one microfluidic channel, whereinthe at least one array comprises a plurality of biofunctionalizednanometer-scale mechanical resonators each having at least one detectorin signal communication therewith for measuring the resonance motion ofthe resonator.
 34. A molecular detector system as described in claim 33,wherein the plurality of resonators has at least two differentbiofunctionalizations.
 35. A molecular detector system as described inclaim 33, wherein said nanometer-scale mechanical resonators contain atleast one first resonator that is biofunctionalized with a firstreceptor or ligand and at least one second resonator that isbiofunctionalized with a second receptor or ligand.
 36. A moleculardetector system as described in claim 33, wherein the at least one arrayof molecular detector devices is functionalized to monitor for a singlesubstance.
 37. A molecular detector system as described in claim 33,wherein the at least one array of molecular detector devices isfunctionalized so that multiple substances can be identified andmonitored simultaneously.
 38. A molecular detector system as describedin claim 33, wherein the at least one array of molecular detectordevices comprises multiple coupled resonators with a receptorbiofunctionalization on one first resonator and a ligandbiofunctionalization on one second resonator such that the motion of thefirst and second resonators is coupled through the ligand/receptorbiofunctionalization and such that the motion of both resonators ismonitored simultaneously.
 39. A molecular detector system as describedin claim 33, wherein the at least one array of molecular detectordevices comprises at least two different resonators: a driver resonatorand a follower resonator wherein a receptor biofunctionalization isprovided on the driver resonator and a ligand biofunctionalization isprovided on the adjacent follower resonator such that the motion of thedriver and adjacent follower resonators are coupled through theligand/receptor biofunctionalization and such that the motion of bothdriver resonator and adjacent follower resonator is monitoredsimultaneously.
 40. A molecular detector system as described in claim33, wherein the at least one array of molecular detector devicescomprises at least three different resonators: a (+) driver resonator, a(−) driver resonator and a follower resonator, wherein a receptorbiofunctionalization is provided on at least one of the (+) driverresonators and a ligand biofunctionalization on the adjacent followerresonator such that the motion of the (+) driver and adjacent followerresonators is coupled through the ligand/receptor biofunctionalizationand such that the motion of the driver and adjacent follower resonatoris simultaneously monitored.
 41. The molecular detector system asdescribed in claim 40, wherein the (−) driver resonator operates inantiphase to the (+) driver resonator.